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TaKaRa exonucleases iii
Exonucleases Iii, supplied by TaKaRa, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Article Title: Cloning and Sequencing of a Protein Involved in Phagosomal Membrane Fusion in Paramecium
Article Snippet: .. The inserted DNA in the plasmid vectors was treated stepwise with exonucleases III (Takara) and VII ( New England Biolabs ). .. Immediately after alkali denaturation , sequencing of the purified DNA was performed on a double-stranded plasmid by the dideoxynucleotide chain termination method ( ).

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    TaKaRa exonuclease iii
    Analysis of terminal restriction fragments from replicated linear DNAs. (A and B) The bound fraction of pSVO11-bead replication products was purified and treated with either λ exonuclease or exonuclease <t>III.</t> To know the exonuclease digestion rates, we treated a separately prepared 199-bp terminal fragment with these exonucleases and found that under the employed conditions, approximately 100 nt is digested from the ends, albeit relatively asymmetrically (data not shown). After the digestion, a half aliquot of the <t>DNA</t> was further treated with Dra I, which produces 199- and 497-bp fragments from the left and right arms of the DNA, respectively (A). Samples were run in a 6% denaturing acrylamide gel, dried, and autoradiographed. Heavily and lightly exposed autoradiographs of the same gel are shown. Control pSVO11 DNA was digested with Bsr FI, and the two ends were filled-in with dNTPs. The resultant blunt-ended linear pSVO11 was first treated with either λ exonuclease or exonuclease III, followed by Dra I digestion. The products were first dephosphorylated by alkaline phosphatase at their 5′ ends and then labeled by T4 polynucleotide kinase and [γ- 32 P]ATP. Dra I digests DNA at a TTT/AAA site, leaving blunt ends. Therefore, the two 199-nt and 497-nt fragment strands have the same nucleotide lengths (arrows). However, because of the effect of different base compositions on migration rates, two distinct 199-nt single-stranded DNA bands are visible in lane 1. The upper and lower bands (marked by open and filled circles, respectively) of the 199-nt doublet were completely digested by λ exonuclease and exonuclease III, respectively (lanes 2 to 5). The 199- and 197-nt bands were detected in pSV011-band replication products. These two bands were resistant to λ exonuclease (lanes 9 and 11). In contrast, the 199-nt band was completely digested, and the 497-nt band was significantly trimmed by exonuclease III (lanes 13 and 15; shorter-sized 497-nt bands are indicated by a bracket). These results indicate that the observed 497- and 199-nt bands were derived solely from a strand whose 3′ ends correspond to nascent radiolabeled DNA ends. Several extra bands were observed in lane 7. We do not know the precise origin of these signals. However, because they are both λ exonuclease and exonuclease III sensitive, it is likely they represent unligated lagging strand DNA molecules derived from internal template regions. It seemed that λ exonuclease had reached the Dra I site on the template (cold) strand of some molecules, because the signal intensity of the 199-nt band decreased after the λ exonuclease treatment.
    Exonuclease Iii, supplied by TaKaRa, used in various techniques. Bioz Stars score: 93/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/exonuclease iii/product/TaKaRa
    Average 93 stars, based on 8 article reviews
    Price from $9.99 to $1999.99
    exonuclease iii - by Bioz Stars, 2020-07
    93/100 stars
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    Analysis of terminal restriction fragments from replicated linear DNAs. (A and B) The bound fraction of pSVO11-bead replication products was purified and treated with either λ exonuclease or exonuclease III. To know the exonuclease digestion rates, we treated a separately prepared 199-bp terminal fragment with these exonucleases and found that under the employed conditions, approximately 100 nt is digested from the ends, albeit relatively asymmetrically (data not shown). After the digestion, a half aliquot of the DNA was further treated with Dra I, which produces 199- and 497-bp fragments from the left and right arms of the DNA, respectively (A). Samples were run in a 6% denaturing acrylamide gel, dried, and autoradiographed. Heavily and lightly exposed autoradiographs of the same gel are shown. Control pSVO11 DNA was digested with Bsr FI, and the two ends were filled-in with dNTPs. The resultant blunt-ended linear pSVO11 was first treated with either λ exonuclease or exonuclease III, followed by Dra I digestion. The products were first dephosphorylated by alkaline phosphatase at their 5′ ends and then labeled by T4 polynucleotide kinase and [γ- 32 P]ATP. Dra I digests DNA at a TTT/AAA site, leaving blunt ends. Therefore, the two 199-nt and 497-nt fragment strands have the same nucleotide lengths (arrows). However, because of the effect of different base compositions on migration rates, two distinct 199-nt single-stranded DNA bands are visible in lane 1. The upper and lower bands (marked by open and filled circles, respectively) of the 199-nt doublet were completely digested by λ exonuclease and exonuclease III, respectively (lanes 2 to 5). The 199- and 197-nt bands were detected in pSV011-band replication products. These two bands were resistant to λ exonuclease (lanes 9 and 11). In contrast, the 199-nt band was completely digested, and the 497-nt band was significantly trimmed by exonuclease III (lanes 13 and 15; shorter-sized 497-nt bands are indicated by a bracket). These results indicate that the observed 497- and 199-nt bands were derived solely from a strand whose 3′ ends correspond to nascent radiolabeled DNA ends. Several extra bands were observed in lane 7. We do not know the precise origin of these signals. However, because they are both λ exonuclease and exonuclease III sensitive, it is likely they represent unligated lagging strand DNA molecules derived from internal template regions. It seemed that λ exonuclease had reached the Dra I site on the template (cold) strand of some molecules, because the signal intensity of the 199-nt band decreased after the λ exonuclease treatment.

    Journal: Molecular and Cellular Biology

    Article Title: In Vitro Reconstitution of the End Replication Problem

    doi: 10.1128/MCB.21.17.5753-5766.2001

    Figure Lengend Snippet: Analysis of terminal restriction fragments from replicated linear DNAs. (A and B) The bound fraction of pSVO11-bead replication products was purified and treated with either λ exonuclease or exonuclease III. To know the exonuclease digestion rates, we treated a separately prepared 199-bp terminal fragment with these exonucleases and found that under the employed conditions, approximately 100 nt is digested from the ends, albeit relatively asymmetrically (data not shown). After the digestion, a half aliquot of the DNA was further treated with Dra I, which produces 199- and 497-bp fragments from the left and right arms of the DNA, respectively (A). Samples were run in a 6% denaturing acrylamide gel, dried, and autoradiographed. Heavily and lightly exposed autoradiographs of the same gel are shown. Control pSVO11 DNA was digested with Bsr FI, and the two ends were filled-in with dNTPs. The resultant blunt-ended linear pSVO11 was first treated with either λ exonuclease or exonuclease III, followed by Dra I digestion. The products were first dephosphorylated by alkaline phosphatase at their 5′ ends and then labeled by T4 polynucleotide kinase and [γ- 32 P]ATP. Dra I digests DNA at a TTT/AAA site, leaving blunt ends. Therefore, the two 199-nt and 497-nt fragment strands have the same nucleotide lengths (arrows). However, because of the effect of different base compositions on migration rates, two distinct 199-nt single-stranded DNA bands are visible in lane 1. The upper and lower bands (marked by open and filled circles, respectively) of the 199-nt doublet were completely digested by λ exonuclease and exonuclease III, respectively (lanes 2 to 5). The 199- and 197-nt bands were detected in pSV011-band replication products. These two bands were resistant to λ exonuclease (lanes 9 and 11). In contrast, the 199-nt band was completely digested, and the 497-nt band was significantly trimmed by exonuclease III (lanes 13 and 15; shorter-sized 497-nt bands are indicated by a bracket). These results indicate that the observed 497- and 199-nt bands were derived solely from a strand whose 3′ ends correspond to nascent radiolabeled DNA ends. Several extra bands were observed in lane 7. We do not know the precise origin of these signals. However, because they are both λ exonuclease and exonuclease III sensitive, it is likely they represent unligated lagging strand DNA molecules derived from internal template regions. It seemed that λ exonuclease had reached the Dra I site on the template (cold) strand of some molecules, because the signal intensity of the 199-nt band decreased after the λ exonuclease treatment.

    Article Snippet: The purified DNA was either treated with or without λ exonuclease (GIBCO), exonuclease III (Takara), and exonuclease I (New England BioLabs).

    Techniques: Purification, Acrylamide Gel Assay, Labeling, Migration, Derivative Assay

    Amount of residual DNAs in the presence of 0% (w/v) (open circles), 5% (w/v) (closed circles), 10% (w/v) (closed triangles), 15% (w/v) (closed squares) and 20% (w/v) (closed diamonds) of PEG and ( A ) 0.1 U DNase I, ( B ) 0.15 U S1 nuclease, ( C ) 1 U exonuclease III (inset shows 10 U), ( D ) 0.5 U exonuclease I (inset shows 5 U). A ssDNA was used as a substrate for S1 nuclease and exonuclease I and a dsDNA was used as a substrate for DNase I and exonuclease III. PEG 4000 was used as the crowding agent for DNase I and S1 nuclease reactions, and PEG 8000 was used for exonucleases III and I. Error bars (smaller than ±2%) were omitted for clarity.

    Journal: Nucleic Acids Research

    Article Title: Regulation of DNA nucleases by molecular crowding

    doi: 10.1093/nar/gkm445

    Figure Lengend Snippet: Amount of residual DNAs in the presence of 0% (w/v) (open circles), 5% (w/v) (closed circles), 10% (w/v) (closed triangles), 15% (w/v) (closed squares) and 20% (w/v) (closed diamonds) of PEG and ( A ) 0.1 U DNase I, ( B ) 0.15 U S1 nuclease, ( C ) 1 U exonuclease III (inset shows 10 U), ( D ) 0.5 U exonuclease I (inset shows 5 U). A ssDNA was used as a substrate for S1 nuclease and exonuclease I and a dsDNA was used as a substrate for DNase I and exonuclease III. PEG 4000 was used as the crowding agent for DNase I and S1 nuclease reactions, and PEG 8000 was used for exonucleases III and I. Error bars (smaller than ±2%) were omitted for clarity.

    Article Snippet: Exonuclease I from Escherichia coli , S1 nuclease from Asergillus oryzae and exonuclease III from E. coli were purchased from Takara Bio (Tokyo, Japan).

    Techniques:

    Exosomes secretion prevents ATM/ATR-dependent DDR. Pre-senescent TIG-3 cells were transfected with two different sets of validated siRNA oligos indicated at the top of the panel for twice at 2 day intervals. These cells were then subjected to western blotting using antibodies shown right ( a ) or to cell proliferation analysis ( b ). Tubulin was used as a loading control ( a ). The representative data from three independent experiments are shown. Error bars indicate mean±s.d. of triplicate measurements.

    Journal: Nature Communications

    Article Title: Exosomes maintain cellular homeostasis by excreting harmful DNA from cells

    doi: 10.1038/ncomms15287

    Figure Lengend Snippet: Exosomes secretion prevents ATM/ATR-dependent DDR. Pre-senescent TIG-3 cells were transfected with two different sets of validated siRNA oligos indicated at the top of the panel for twice at 2 day intervals. These cells were then subjected to western blotting using antibodies shown right ( a ) or to cell proliferation analysis ( b ). Tubulin was used as a loading control ( a ). The representative data from three independent experiments are shown. Error bars indicate mean±s.d. of triplicate measurements.

    Article Snippet: Quantitative measurement of isolated exosomal DNA To reduce external DNA contamination, prior to DNA extraction, exosomes were treated with DNase I (Roche Inc.) and Exonuclease III (Takara Inc.), according to the manufacturers' instructions .

    Techniques: Transfection, Western Blot

    Inhibition of exosome secretion in mouse liver. ICR mice were subjected to hydrodynamic tail vein injection with plasmid encoding firefly luciferase or small hairpin RNA (shRNA) against Alix or control ( n =3 per group). After 48 h, the mice transfected with firefly luciferase were subjected to i n vivo bioluminescent imaging for confirmation of the transfection efficiency ( a ), and then other mice were euthanized and livers were subjected to western blotting using antibodies shown right ( b ), NanoSight analysis (NTA) for quantitative measurement of isolated exosome particles ( c ) or to immunofluorescence analysis of liver section ( d ). Tubulin was used as a loading control ( b ). Section of livers were subjected to immunofluorescence staining for markers of DNA damage (53BP1 (red) and 4′,6-diamidino-2-phenylindole (blue)) ( d ). The histograms indicate the percentage of nuclei that contain more than 3 foci positive for 53BP1 staining. At least 100 cells were scored per group. The representative data from three independent experiments are shown. For all graphs, error bars indicate mean±s.d. of triplicate measurements. (** P

    Journal: Nature Communications

    Article Title: Exosomes maintain cellular homeostasis by excreting harmful DNA from cells

    doi: 10.1038/ncomms15287

    Figure Lengend Snippet: Inhibition of exosome secretion in mouse liver. ICR mice were subjected to hydrodynamic tail vein injection with plasmid encoding firefly luciferase or small hairpin RNA (shRNA) against Alix or control ( n =3 per group). After 48 h, the mice transfected with firefly luciferase were subjected to i n vivo bioluminescent imaging for confirmation of the transfection efficiency ( a ), and then other mice were euthanized and livers were subjected to western blotting using antibodies shown right ( b ), NanoSight analysis (NTA) for quantitative measurement of isolated exosome particles ( c ) or to immunofluorescence analysis of liver section ( d ). Tubulin was used as a loading control ( b ). Section of livers were subjected to immunofluorescence staining for markers of DNA damage (53BP1 (red) and 4′,6-diamidino-2-phenylindole (blue)) ( d ). The histograms indicate the percentage of nuclei that contain more than 3 foci positive for 53BP1 staining. At least 100 cells were scored per group. The representative data from three independent experiments are shown. For all graphs, error bars indicate mean±s.d. of triplicate measurements. (** P

    Article Snippet: Quantitative measurement of isolated exosomal DNA To reduce external DNA contamination, prior to DNA extraction, exosomes were treated with DNase I (Roche Inc.) and Exonuclease III (Takara Inc.), according to the manufacturers' instructions .

    Techniques: Inhibition, Mouse Assay, Injection, Plasmid Preparation, Luciferase, shRNA, Transfection, Imaging, Western Blot, Isolation, Immunofluorescence, Staining

    Exosome secretion prevents viral hijacking of cellular machinery. ( a ) Timeline of the experimental procedure. ( b – e ) Pre-senescent TIG-3 cells transfected with indicated siRNA oligos followed by infection with recombinant adenovirus encoding GFP (Ad-GFP) were subjected to western blotting using antibodies shown right ( b ), NanoSight analysis (NTA) and western blotting against canonical exosome markers for quantitative measurement of isolated exosome particles ( c ), quantitative measurement of isolated adenoviral DNA from exosome using quantitative PCR ( d ), or to microscopic analysis of GFP expression ( e ). The representative data from three independent experiments are shown. ( f ) Timeline of the experimental procedure. ( g – i ) 293 cells were transfected with indicated siRNA oligos followed by infection with Ad-GFP. These cells were then subjected to western blotting using antibodies shown right ( g ), NanoSight analysis (NTA) and western blotting against canonical exosome markers for quantitative measurement of isolated exosome particles ( h ) or to titration of generated Ad-GFP ( i ). The histograms indicate the virus titre ( i ). For all graphs, error bars indicate mean±s.d. of triplicate measurements. (** P

    Journal: Nature Communications

    Article Title: Exosomes maintain cellular homeostasis by excreting harmful DNA from cells

    doi: 10.1038/ncomms15287

    Figure Lengend Snippet: Exosome secretion prevents viral hijacking of cellular machinery. ( a ) Timeline of the experimental procedure. ( b – e ) Pre-senescent TIG-3 cells transfected with indicated siRNA oligos followed by infection with recombinant adenovirus encoding GFP (Ad-GFP) were subjected to western blotting using antibodies shown right ( b ), NanoSight analysis (NTA) and western blotting against canonical exosome markers for quantitative measurement of isolated exosome particles ( c ), quantitative measurement of isolated adenoviral DNA from exosome using quantitative PCR ( d ), or to microscopic analysis of GFP expression ( e ). The representative data from three independent experiments are shown. ( f ) Timeline of the experimental procedure. ( g – i ) 293 cells were transfected with indicated siRNA oligos followed by infection with Ad-GFP. These cells were then subjected to western blotting using antibodies shown right ( g ), NanoSight analysis (NTA) and western blotting against canonical exosome markers for quantitative measurement of isolated exosome particles ( h ) or to titration of generated Ad-GFP ( i ). The histograms indicate the virus titre ( i ). For all graphs, error bars indicate mean±s.d. of triplicate measurements. (** P

    Article Snippet: Quantitative measurement of isolated exosomal DNA To reduce external DNA contamination, prior to DNA extraction, exosomes were treated with DNase I (Roche Inc.) and Exonuclease III (Takara Inc.), according to the manufacturers' instructions .

    Techniques: Transfection, Infection, Recombinant, Western Blot, Isolation, Real-time Polymerase Chain Reaction, Expressing, Titration, Generated

    Inhibition of exosome secretion in pre-senescent HDFs. ( a ) Pre-senescent TIG-3 cells were subjected to transfection with indicated siRNA oligos twice (at 2 day intervals). These cells were then subjected to western blotting using antibodies shown right (WCL) or to exosome isolation followed by western blotting using antibodies against canonical exosome markers shown right (exosome) and NanoSight analysis (NTA) for quantitative measurement of isolated exosome particles. The representative data from three independent experiments are shown. Tubulin was used as a loading control. ( b – d ) Pre-senescent TIG-3 cells cultured under the conditions described in a were subjected to cell proliferation analysis ( b ), apoptosis analysis at day 4 ( c ) or to immunofluorescence staining for markers of DNA damage (γ-H2AX [red], phosphor-Ser/Thr ATM/ATR (pST/Q) substrate [green] and 4′,6-diamidino-2-phenylindole [blue]) ( d ). The representative data from three independent experiments are shown. The histograms indicate the percentage of nuclei that contain more than 3 foci positive for both γ-H2AX and pST/Q staining ( d ). At least 100 cells were scored per group ( d ). ( e , f ) Pre-senescent TIG-3 cells were infected with retrovirus encoding flag-tagged wild-type Alix or Rab27a protein containing a mutated siRNA cleavage site (lanes 3 and 4) or empty vector (lanes 1 and 2). After selection with puromycin, cells were transfected with indicated siRNA oligos and then subjected to western blotting using antibodies shown right, NanoSight analysis for quantitative measurement of isolated exosome particles, apoptosis analysis at day 4 or to immunofluorescence staining for markers of DNA damage. Tubulin was used as a loading control. The representative data from three independent experiments are shown. For all graphs, error bars indicate mean±s.d. of triplicate measurements. (** P

    Journal: Nature Communications

    Article Title: Exosomes maintain cellular homeostasis by excreting harmful DNA from cells

    doi: 10.1038/ncomms15287

    Figure Lengend Snippet: Inhibition of exosome secretion in pre-senescent HDFs. ( a ) Pre-senescent TIG-3 cells were subjected to transfection with indicated siRNA oligos twice (at 2 day intervals). These cells were then subjected to western blotting using antibodies shown right (WCL) or to exosome isolation followed by western blotting using antibodies against canonical exosome markers shown right (exosome) and NanoSight analysis (NTA) for quantitative measurement of isolated exosome particles. The representative data from three independent experiments are shown. Tubulin was used as a loading control. ( b – d ) Pre-senescent TIG-3 cells cultured under the conditions described in a were subjected to cell proliferation analysis ( b ), apoptosis analysis at day 4 ( c ) or to immunofluorescence staining for markers of DNA damage (γ-H2AX [red], phosphor-Ser/Thr ATM/ATR (pST/Q) substrate [green] and 4′,6-diamidino-2-phenylindole [blue]) ( d ). The representative data from three independent experiments are shown. The histograms indicate the percentage of nuclei that contain more than 3 foci positive for both γ-H2AX and pST/Q staining ( d ). At least 100 cells were scored per group ( d ). ( e , f ) Pre-senescent TIG-3 cells were infected with retrovirus encoding flag-tagged wild-type Alix or Rab27a protein containing a mutated siRNA cleavage site (lanes 3 and 4) or empty vector (lanes 1 and 2). After selection with puromycin, cells were transfected with indicated siRNA oligos and then subjected to western blotting using antibodies shown right, NanoSight analysis for quantitative measurement of isolated exosome particles, apoptosis analysis at day 4 or to immunofluorescence staining for markers of DNA damage. Tubulin was used as a loading control. The representative data from three independent experiments are shown. For all graphs, error bars indicate mean±s.d. of triplicate measurements. (** P

    Article Snippet: Quantitative measurement of isolated exosomal DNA To reduce external DNA contamination, prior to DNA extraction, exosomes were treated with DNase I (Roche Inc.) and Exonuclease III (Takara Inc.), according to the manufacturers' instructions .

    Techniques: Inhibition, Transfection, Western Blot, Isolation, Cell Culture, Immunofluorescence, Staining, Infection, Plasmid Preparation, Selection

    Overexpression of Dnase2a attenuated the effects of Alix or Rab27a knockdown in HDFs. Pre-senescent TIG-3 cells were infected with retrovirus encoding flag-tagged Dnase2a (lanes 4–6) or empty vector (lanes 1–3). After selection with puromycin, cells were transfected with indicated siRNA oligos and then subjected to western blotting using antibodies shown right ( a ), NanoSight analysis (NTA) for quantitative measurement of isolated exosome particles and western blotting using antibodies against canonical exosome markers shown right (exosome) ( b ), isolation of cytoplasmic fraction followed by quantitative PCR (qPCR) analysis of chromosomal DNA ( c ), immunofluorescence staining for markers of DNA damage (γ-H2AX [red], pST/Q (green) and 4′,6-diamidino-2-phenylindole (blue)) ( d ), qPCR analysis of IFNβ gene expression ( e ), analysis of intracellular ROS levels ( e ) or to apoptosis analysis at day 4 ( e ). Tubulin was used as a loading control ( a ). The histograms indicate the percentage of nuclei that contain more than 3 foci positive for both γ-H2AX and pST/Q staining ( d ). At least 100 cells were scored per group ( d ). The representative data from three independent experiments are shown. For all graphs, error bars indicate mean±s.d. of triplicate measurements. (** P

    Journal: Nature Communications

    Article Title: Exosomes maintain cellular homeostasis by excreting harmful DNA from cells

    doi: 10.1038/ncomms15287

    Figure Lengend Snippet: Overexpression of Dnase2a attenuated the effects of Alix or Rab27a knockdown in HDFs. Pre-senescent TIG-3 cells were infected with retrovirus encoding flag-tagged Dnase2a (lanes 4–6) or empty vector (lanes 1–3). After selection with puromycin, cells were transfected with indicated siRNA oligos and then subjected to western blotting using antibodies shown right ( a ), NanoSight analysis (NTA) for quantitative measurement of isolated exosome particles and western blotting using antibodies against canonical exosome markers shown right (exosome) ( b ), isolation of cytoplasmic fraction followed by quantitative PCR (qPCR) analysis of chromosomal DNA ( c ), immunofluorescence staining for markers of DNA damage (γ-H2AX [red], pST/Q (green) and 4′,6-diamidino-2-phenylindole (blue)) ( d ), qPCR analysis of IFNβ gene expression ( e ), analysis of intracellular ROS levels ( e ) or to apoptosis analysis at day 4 ( e ). Tubulin was used as a loading control ( a ). The histograms indicate the percentage of nuclei that contain more than 3 foci positive for both γ-H2AX and pST/Q staining ( d ). At least 100 cells were scored per group ( d ). The representative data from three independent experiments are shown. For all graphs, error bars indicate mean±s.d. of triplicate measurements. (** P

    Article Snippet: Quantitative measurement of isolated exosomal DNA To reduce external DNA contamination, prior to DNA extraction, exosomes were treated with DNase I (Roche Inc.) and Exonuclease III (Takara Inc.), according to the manufacturers' instructions .

    Techniques: Over Expression, Infection, Plasmid Preparation, Selection, Transfection, Western Blot, Isolation, Real-time Polymerase Chain Reaction, Immunofluorescence, Staining, Expressing

    Reduction of ROS levels attenuated the effects of Alix or Rab27a knockdown in HDFs. Pre-senescent TIG-3 cells were transfected with validated siRNA oligos indicated at the top of the panel for two times at 2 day intervals in the presence or absence of 1 mM N -acetyl cysteine. These cells were then subjected to western blotting using antibodies shown right ( a ), analysis of intracellular ROS levels ( b ), immunofluorescence staining for markers of DNA damage (γ-H2AX (red), pST/Q (green) and 4′,6-diamidino-2-phenylindole (blue)) ( c ) or to apoptosis analysis ( d ). The histograms indicate the percentage of nuclei that contain more than 3 foci positive for both γ-H2AX and pST/Q staining ( c ). At least 100 cells were scored per group ( c ). The representative data from three independent experiments are shown. For all graphs, error bars indicate mean±s.d. of triplicate measurements. (* P

    Journal: Nature Communications

    Article Title: Exosomes maintain cellular homeostasis by excreting harmful DNA from cells

    doi: 10.1038/ncomms15287

    Figure Lengend Snippet: Reduction of ROS levels attenuated the effects of Alix or Rab27a knockdown in HDFs. Pre-senescent TIG-3 cells were transfected with validated siRNA oligos indicated at the top of the panel for two times at 2 day intervals in the presence or absence of 1 mM N -acetyl cysteine. These cells were then subjected to western blotting using antibodies shown right ( a ), analysis of intracellular ROS levels ( b ), immunofluorescence staining for markers of DNA damage (γ-H2AX (red), pST/Q (green) and 4′,6-diamidino-2-phenylindole (blue)) ( c ) or to apoptosis analysis ( d ). The histograms indicate the percentage of nuclei that contain more than 3 foci positive for both γ-H2AX and pST/Q staining ( c ). At least 100 cells were scored per group ( c ). The representative data from three independent experiments are shown. For all graphs, error bars indicate mean±s.d. of triplicate measurements. (* P

    Article Snippet: Quantitative measurement of isolated exosomal DNA To reduce external DNA contamination, prior to DNA extraction, exosomes were treated with DNase I (Roche Inc.) and Exonuclease III (Takara Inc.), according to the manufacturers' instructions .

    Techniques: Transfection, Western Blot, Immunofluorescence, Staining

    ssDNA-to-dsDNA Conversion Establishes Stable DNA-DNA Cohesion (A) Time course of second-DNA capture in a protocol 2 assay, comparing WT and 1B3B cohesin. The percentage of free dsDNA captured on ssDNA beads is plotted over time. (B) Same as (A), but the second-DNA capture incubation proceeded for 5 or 30 min in the absence or presence of an ATP-regenerating system (ATP-RG). The indicated additions were made 5 min into the second-DNA capture incubation. (C) Schematic of the experiment to convert ssDNA-to-dsDNA following second-DNA capture to test stabilization against NaCl and EDTA treatment. The gel image shows input and the recovered and released DNAs at the indicated stages of the experiment. The means and standard deviations from three independent experiments are shown in each panel.

    Journal: Cell

    Article Title: Establishment of DNA-DNA Interactions by the Cohesin Ring

    doi: 10.1016/j.cell.2017.12.021

    Figure Lengend Snippet: ssDNA-to-dsDNA Conversion Establishes Stable DNA-DNA Cohesion (A) Time course of second-DNA capture in a protocol 2 assay, comparing WT and 1B3B cohesin. The percentage of free dsDNA captured on ssDNA beads is plotted over time. (B) Same as (A), but the second-DNA capture incubation proceeded for 5 or 30 min in the absence or presence of an ATP-regenerating system (ATP-RG). The indicated additions were made 5 min into the second-DNA capture incubation. (C) Schematic of the experiment to convert ssDNA-to-dsDNA following second-DNA capture to test stabilization against NaCl and EDTA treatment. The gel image shows input and the recovered and released DNAs at the indicated stages of the experiment. The means and standard deviations from three independent experiments are shown in each panel.

    Article Snippet: Escherichia coli exonuclease I, exonuclease III, T4 DNA polymerase (TaKaRa Bio), AcTEV protease (Thermo Fisher Scientific) and PreScission protease (GE Healthcare) were purchased from the indicated manufacturers.

    Techniques: Incubation

    Acetyl-Acceptor Lysines and the Cohesin Loader Promote Second-DNA Capture (A) Protocol 2 experiments were carried out with the indicated order of additions, demonstrating a strong preference of reaction order during second-DNA capture. (B) Protocol 1B was used to test the ability of the indicated cohesin loading cofactors to promote second-DNA capture. Recovered DNA was analyzed by agarose gel electrophoresis and quantified. (C) WT and Psm3 K106Q (KQ) cohesin was used in a protocol 2 experiment. An aliquot was taken after the first dsDNA loading incubation to confirm comparable levels of loading by carrying out the reaction in low-salt condition (15 mM NaCl) before performing second-DNA capture on ssDNA beads followed by agarose gel electrophoresis and quantification. The means and standard deviations from three independent experiments are shown in each panel. See also Figure S4 for experiments that confirm the Mis4 requirement for second-DNA capture and the contribution of the acetyl-acceptor lysines to cohesin loading onto ssDNA.

    Journal: Cell

    Article Title: Establishment of DNA-DNA Interactions by the Cohesin Ring

    doi: 10.1016/j.cell.2017.12.021

    Figure Lengend Snippet: Acetyl-Acceptor Lysines and the Cohesin Loader Promote Second-DNA Capture (A) Protocol 2 experiments were carried out with the indicated order of additions, demonstrating a strong preference of reaction order during second-DNA capture. (B) Protocol 1B was used to test the ability of the indicated cohesin loading cofactors to promote second-DNA capture. Recovered DNA was analyzed by agarose gel electrophoresis and quantified. (C) WT and Psm3 K106Q (KQ) cohesin was used in a protocol 2 experiment. An aliquot was taken after the first dsDNA loading incubation to confirm comparable levels of loading by carrying out the reaction in low-salt condition (15 mM NaCl) before performing second-DNA capture on ssDNA beads followed by agarose gel electrophoresis and quantification. The means and standard deviations from three independent experiments are shown in each panel. See also Figure S4 for experiments that confirm the Mis4 requirement for second-DNA capture and the contribution of the acetyl-acceptor lysines to cohesin loading onto ssDNA.

    Article Snippet: Escherichia coli exonuclease I, exonuclease III, T4 DNA polymerase (TaKaRa Bio), AcTEV protease (Thermo Fisher Scientific) and PreScission protease (GE Healthcare) were purchased from the indicated manufacturers.

    Techniques: Agarose Gel Electrophoresis, Incubation

    Mis4-Ssl3, but not Pds5-Wapl, Promote Second-DNA Capture, Related to Figure 5 (A) Protocol 1B reactions were initiated by cohesin and Mis4-Ssl3. Then the dsDNA beads were washed and increasing concentrations of Pds5-Wapl were included for second-DNA capture. A reaction in which Mis4-Ssl3 was added back is included for comparison. The graph shows quantification of recovered free ssDNA detected by agarose gel electrophoresis. (B) Protocol 1B reactions were carried out in the presence of the indicated loading cofactors as described in Figure 5 B, but 1B3B cohesin was used. The graph shows means and the range of recovered ssDNA from two independent experiments. (C) Protocol 2 reactions were carried out in the presence of the indicated loading cofactors. The gel image shows recovery of free dsDNA on the ssDNA beads, the graph reports means and standard deviations from three independent experiments. (D) Acetyl-acceptor lysines on Psm3 contribute to ssDNA loading. DNA loading reactions were carried out with the indicated cohesin complexes using dsDNA or ssDNA as substrates. The graph shows means and standard deviations of the recovered DNA from three independent experiments.

    Journal: Cell

    Article Title: Establishment of DNA-DNA Interactions by the Cohesin Ring

    doi: 10.1016/j.cell.2017.12.021

    Figure Lengend Snippet: Mis4-Ssl3, but not Pds5-Wapl, Promote Second-DNA Capture, Related to Figure 5 (A) Protocol 1B reactions were initiated by cohesin and Mis4-Ssl3. Then the dsDNA beads were washed and increasing concentrations of Pds5-Wapl were included for second-DNA capture. A reaction in which Mis4-Ssl3 was added back is included for comparison. The graph shows quantification of recovered free ssDNA detected by agarose gel electrophoresis. (B) Protocol 1B reactions were carried out in the presence of the indicated loading cofactors as described in Figure 5 B, but 1B3B cohesin was used. The graph shows means and the range of recovered ssDNA from two independent experiments. (C) Protocol 2 reactions were carried out in the presence of the indicated loading cofactors. The gel image shows recovery of free dsDNA on the ssDNA beads, the graph reports means and standard deviations from three independent experiments. (D) Acetyl-acceptor lysines on Psm3 contribute to ssDNA loading. DNA loading reactions were carried out with the indicated cohesin complexes using dsDNA or ssDNA as substrates. The graph shows means and standard deviations of the recovered DNA from three independent experiments.

    Article Snippet: Escherichia coli exonuclease I, exonuclease III, T4 DNA polymerase (TaKaRa Bio), AcTEV protease (Thermo Fisher Scientific) and PreScission protease (GE Healthcare) were purchased from the indicated manufacturers.

    Techniques: Agarose Gel Electrophoresis

    RPA Impacts on Sister Chromatid Cohesion Establishment In Vivo (A) Effect of the rfa1 G77E mutation or RPA overexpression (RPA OE) on sister chromatid cohesion in ctf18Δ cells. Cells were synchronized and arrested in mitosis by nocodazole treatment. Sister chromatid cohesion at the GFP-marked URA3 locus was analyzed. Western blotting confirmed RPA overexpression. At least 100 cells were scored under each condition. The graph shows means and standard deviations from three independent experiments. (B) Smc3 acetylation was analyzed in synchronized cultures from the strains above by western blotting. The acetyl-Smc3 signal, normalized to tubulin and then to the WT signal at 90 min, was quantified in three independent repeats of the experiment. The means and standard deviations are shown. (C) The cohesin loader promotes sister chromatid cohesion establishment. Sister chromatid cohesion was monitored at indicated time points following release from G1 or HU under the indicated conditions and genotypes. (D) A model for the establishment of sister chromatid cohesion at the DNA replication fork. See the Discussion for details. See also Figure S6 for supporting genetic and cell-cycle analyses that explore the role of RPA in sister chromatid cohesion establishment.

    Journal: Cell

    Article Title: Establishment of DNA-DNA Interactions by the Cohesin Ring

    doi: 10.1016/j.cell.2017.12.021

    Figure Lengend Snippet: RPA Impacts on Sister Chromatid Cohesion Establishment In Vivo (A) Effect of the rfa1 G77E mutation or RPA overexpression (RPA OE) on sister chromatid cohesion in ctf18Δ cells. Cells were synchronized and arrested in mitosis by nocodazole treatment. Sister chromatid cohesion at the GFP-marked URA3 locus was analyzed. Western blotting confirmed RPA overexpression. At least 100 cells were scored under each condition. The graph shows means and standard deviations from three independent experiments. (B) Smc3 acetylation was analyzed in synchronized cultures from the strains above by western blotting. The acetyl-Smc3 signal, normalized to tubulin and then to the WT signal at 90 min, was quantified in three independent repeats of the experiment. The means and standard deviations are shown. (C) The cohesin loader promotes sister chromatid cohesion establishment. Sister chromatid cohesion was monitored at indicated time points following release from G1 or HU under the indicated conditions and genotypes. (D) A model for the establishment of sister chromatid cohesion at the DNA replication fork. See the Discussion for details. See also Figure S6 for supporting genetic and cell-cycle analyses that explore the role of RPA in sister chromatid cohesion establishment.

    Article Snippet: Escherichia coli exonuclease I, exonuclease III, T4 DNA polymerase (TaKaRa Bio), AcTEV protease (Thermo Fisher Scientific) and PreScission protease (GE Healthcare) were purchased from the indicated manufacturers.

    Techniques: Recombinase Polymerase Amplification, In Vivo, Mutagenesis, Over Expression, Western Blot

    Topological but Labile ssDNA Embrace by the Cohesin Ring (A) Gel images and quantification of cohesin-loading assays using ssDNA or dsDNA as the substrate. Mis4-Ssl3 (MS) or Pds5-Wapl (PW) were added in the presence or absence of ATP. The graph shows the means and standard deviation from three independent experiments. (B) Following loading, the recovered material was challenged with NaCl and EDTA. The gel image and graph show DNA recovery at the indicated stages of the experiment. Means and standard deviations from three independent experiments are given. (C) Schematic and outcome of the dsDNA-to-ssDNA conversion experiment using E. coli exonuclease III (exoIII). Supernatant (S) and beads-bound (B) fractions were analyzed after the NaCl and EDTA chase. The graph indicates means and standard deviations from three independent experiments. (D) Specificity of second-ssDNA capture. Gel images and quantification of the protocol 1B second-DNA capture experiments in the presence of indicated ratio of nicked circular dsDNA competitor. The graph shows the means and standard deviation from three independent experiments. See also Figure S3 , showing ssDNA stimulation of the cohesin ATPase and a control that released ssDNA remains circular.

    Journal: Cell

    Article Title: Establishment of DNA-DNA Interactions by the Cohesin Ring

    doi: 10.1016/j.cell.2017.12.021

    Figure Lengend Snippet: Topological but Labile ssDNA Embrace by the Cohesin Ring (A) Gel images and quantification of cohesin-loading assays using ssDNA or dsDNA as the substrate. Mis4-Ssl3 (MS) or Pds5-Wapl (PW) were added in the presence or absence of ATP. The graph shows the means and standard deviation from three independent experiments. (B) Following loading, the recovered material was challenged with NaCl and EDTA. The gel image and graph show DNA recovery at the indicated stages of the experiment. Means and standard deviations from three independent experiments are given. (C) Schematic and outcome of the dsDNA-to-ssDNA conversion experiment using E. coli exonuclease III (exoIII). Supernatant (S) and beads-bound (B) fractions were analyzed after the NaCl and EDTA chase. The graph indicates means and standard deviations from three independent experiments. (D) Specificity of second-ssDNA capture. Gel images and quantification of the protocol 1B second-DNA capture experiments in the presence of indicated ratio of nicked circular dsDNA competitor. The graph shows the means and standard deviation from three independent experiments. See also Figure S3 , showing ssDNA stimulation of the cohesin ATPase and a control that released ssDNA remains circular.

    Article Snippet: Escherichia coli exonuclease I, exonuclease III, T4 DNA polymerase (TaKaRa Bio), AcTEV protease (Thermo Fisher Scientific) and PreScission protease (GE Healthcare) were purchased from the indicated manufacturers.

    Techniques: Mass Spectrometry, Standard Deviation

    Second-DNA Capture is Topological in Nature, Related to Figure 2 (A) Cohesin must topologically embrace dsDNA to mediate second-DNA capture. Protocol 1 reactions were carried out with ‘closed’ (C) or ‘linear’ (L) topology dsDNA beads. The gel image and graph show recovery of free ssDNA. The graph shows means and standard deviation from three independent experiments (WT cohesin) or the range of recovered ssDNA from two independent experiments (1B3B cohesin). (B) A DNA release experiment as shown in Figure 2 A was carried out using 1B3B cohesin. (C) Schematic of a DNA release experiment following protocol 2 s DNA capture. The ssDNA substrate was converted to dsDNA by DNA synthesis following capture. Then either of the two circular DNAs was digested with unique restriction enzymes, PstI (DNA beads) or BglII (free dsDNA). Recovered DNAs at the indicated stages of the experiment were analyzed by agarose gel electrophoresis. Input, bead bound (B) and supernatant (S) fractions are shown. (D) TEV cleavage of cohesin following second-DNA capture using protocol 2. The gel shows a representative image of input and recovered DNA, using WT and TEV cleavable (21TEV) cohesin, without or with TEV protease (TEV) treatment. After TEV protease treatment, the beads were washed with high salt buffer and recovered DNA was analyzed. The graph depicts the means and standard deviations from three independent experiments.

    Journal: Cell

    Article Title: Establishment of DNA-DNA Interactions by the Cohesin Ring

    doi: 10.1016/j.cell.2017.12.021

    Figure Lengend Snippet: Second-DNA Capture is Topological in Nature, Related to Figure 2 (A) Cohesin must topologically embrace dsDNA to mediate second-DNA capture. Protocol 1 reactions were carried out with ‘closed’ (C) or ‘linear’ (L) topology dsDNA beads. The gel image and graph show recovery of free ssDNA. The graph shows means and standard deviation from three independent experiments (WT cohesin) or the range of recovered ssDNA from two independent experiments (1B3B cohesin). (B) A DNA release experiment as shown in Figure 2 A was carried out using 1B3B cohesin. (C) Schematic of a DNA release experiment following protocol 2 s DNA capture. The ssDNA substrate was converted to dsDNA by DNA synthesis following capture. Then either of the two circular DNAs was digested with unique restriction enzymes, PstI (DNA beads) or BglII (free dsDNA). Recovered DNAs at the indicated stages of the experiment were analyzed by agarose gel electrophoresis. Input, bead bound (B) and supernatant (S) fractions are shown. (D) TEV cleavage of cohesin following second-DNA capture using protocol 2. The gel shows a representative image of input and recovered DNA, using WT and TEV cleavable (21TEV) cohesin, without or with TEV protease (TEV) treatment. After TEV protease treatment, the beads were washed with high salt buffer and recovered DNA was analyzed. The graph depicts the means and standard deviations from three independent experiments.

    Article Snippet: Escherichia coli exonuclease I, exonuclease III, T4 DNA polymerase (TaKaRa Bio), AcTEV protease (Thermo Fisher Scientific) and PreScission protease (GE Healthcare) were purchased from the indicated manufacturers.

    Techniques: Standard Deviation, DNA Synthesis, Agarose Gel Electrophoresis

    ssDNA, but not dsDNA, Is a Substrate for Second-Strand Capture, Related to Figure 1 (A and B) Extended gel images of Figures 1 A and 1B, showing both beads bound and supernatant fractions. 25% of the supernatant fractions are shown. (C) Protocol 2 reactions were carried out using the indicated protein concentrations (each of cohesin, Mis4-Ssl3 and Psc3). The gel image and quantification of captured dsDNA are shown. (D) A typical gel image of inputs and products of a second-DNA capture reaction following protocol 1B, performed with WT cohesin. (E) Gel images showing second-DNA capture by 1B3B cohesin, containing Walker B mutations in both Psm1 and Psm3, using a protocol 1 reaction. (F) As (E), but the reaction followed protocol 2. (G) Competition of ATP with ADP or non-hydrolyzable ATP-γ−S. As in Figure 1 E (top gel image), but an additional reaction was performed in which 0.25 mM of ATP was present in all reactions that were then supplemented by additional nucleotides. The ability of ADP and ATP-γ−S to compete with ATP demonstrates that both nucleotides are able to bind cohesin, but that their hydrolysis is required for second-DNA capture. (H) Cohesin mediates second-DNA capture irrespective of sequence homology. Protocol 2 reactions were carried out using pSKsxAS ssDNA (partially homologous to the free dsDNA substrate) or ΦX174 virion ssDNA (of unrelated sequence) as substrates. The graph presents means and standard deviations from three independent experiments.

    Journal: Cell

    Article Title: Establishment of DNA-DNA Interactions by the Cohesin Ring

    doi: 10.1016/j.cell.2017.12.021

    Figure Lengend Snippet: ssDNA, but not dsDNA, Is a Substrate for Second-Strand Capture, Related to Figure 1 (A and B) Extended gel images of Figures 1 A and 1B, showing both beads bound and supernatant fractions. 25% of the supernatant fractions are shown. (C) Protocol 2 reactions were carried out using the indicated protein concentrations (each of cohesin, Mis4-Ssl3 and Psc3). The gel image and quantification of captured dsDNA are shown. (D) A typical gel image of inputs and products of a second-DNA capture reaction following protocol 1B, performed with WT cohesin. (E) Gel images showing second-DNA capture by 1B3B cohesin, containing Walker B mutations in both Psm1 and Psm3, using a protocol 1 reaction. (F) As (E), but the reaction followed protocol 2. (G) Competition of ATP with ADP or non-hydrolyzable ATP-γ−S. As in Figure 1 E (top gel image), but an additional reaction was performed in which 0.25 mM of ATP was present in all reactions that were then supplemented by additional nucleotides. The ability of ADP and ATP-γ−S to compete with ATP demonstrates that both nucleotides are able to bind cohesin, but that their hydrolysis is required for second-DNA capture. (H) Cohesin mediates second-DNA capture irrespective of sequence homology. Protocol 2 reactions were carried out using pSKsxAS ssDNA (partially homologous to the free dsDNA substrate) or ΦX174 virion ssDNA (of unrelated sequence) as substrates. The graph presents means and standard deviations from three independent experiments.

    Article Snippet: Escherichia coli exonuclease I, exonuclease III, T4 DNA polymerase (TaKaRa Bio), AcTEV protease (Thermo Fisher Scientific) and PreScission protease (GE Healthcare) were purchased from the indicated manufacturers.

    Techniques: Sequencing

    Second-DNA Capture by the Fission Yeast Cohesin Ring (A) Schematic of the second-DNA capture assay (protocol 1) and a gel image showing input and recovered DNA from the assay performed with the indicated substrates. ds, dsDNA; ss, ssDNA; rc, relaxed circular; c, circular. All reactions were carried out in the presence of ATP and an ATP regenerating system. 16.7% or 25% of input free dsDNA or ssDNA are shown. (B) Schematic of the second-DNA capture assay (protocol 2) and a representative gel image. 25% of input DNA is shown. (C and D) Quantification of the assays in (A) and (B), respectively, performed with WT and 1B3B cohesin. The means and standard deviations from three independent experiments are shown. (E) Quantification of second-DNA capture, using protocol 1B, in the absence or presence of the indicated adenosine derivatives. The means and standard deviations from three independent experiments are shown. See also Figure S1 for gel images that include supernatant fractions, reactions using 1B3B cohesin, titration of components, ATP competition, and an assay using ΦX174 ssDNA.

    Journal: Cell

    Article Title: Establishment of DNA-DNA Interactions by the Cohesin Ring

    doi: 10.1016/j.cell.2017.12.021

    Figure Lengend Snippet: Second-DNA Capture by the Fission Yeast Cohesin Ring (A) Schematic of the second-DNA capture assay (protocol 1) and a gel image showing input and recovered DNA from the assay performed with the indicated substrates. ds, dsDNA; ss, ssDNA; rc, relaxed circular; c, circular. All reactions were carried out in the presence of ATP and an ATP regenerating system. 16.7% or 25% of input free dsDNA or ssDNA are shown. (B) Schematic of the second-DNA capture assay (protocol 2) and a representative gel image. 25% of input DNA is shown. (C and D) Quantification of the assays in (A) and (B), respectively, performed with WT and 1B3B cohesin. The means and standard deviations from three independent experiments are shown. (E) Quantification of second-DNA capture, using protocol 1B, in the absence or presence of the indicated adenosine derivatives. The means and standard deviations from three independent experiments are shown. See also Figure S1 for gel images that include supernatant fractions, reactions using 1B3B cohesin, titration of components, ATP competition, and an assay using ΦX174 ssDNA.

    Article Snippet: Escherichia coli exonuclease I, exonuclease III, T4 DNA polymerase (TaKaRa Bio), AcTEV protease (Thermo Fisher Scientific) and PreScission protease (GE Healthcare) were purchased from the indicated manufacturers.

    Techniques: Titration